This invention is directed to the use of 19F NMR spectroscopy to monitor and quantitate solid-phase reactions, and fluorine-containing solid-phase reagents useful therefor.
Solid-phase synthetic techniques, in which a reagent is immobilized on a polymeric material, which material is inert to the reagents and reaction conditions. employed, as well as being insoluble in the media used, are important synthetic tools for preparing amides and peptides as well as for effecting various functional group transformations. For solid-phase peptide synthesis, a summary of the many techniques employed may be found in J. M. Stewart and J. D. Young, Solid-phase Peptide Synthesis, 2nd. Ed., Pierce Chemical Co. (Chicago, Ill., 1984); J. Meienhofer, Hormonal Proteins and Peptides, vol. 2, p. 46, Academic Press (New York), 1973; and E. Atherton and R. C. Sheppard, Solid-phase Peptide Synthesis: A Practical Approach, IRL Press at Oxford University Press (Oxford, 1989). For the use of solid-phase methodology in the preparation of non-peptide molecules see Leznoff, C. C., Acc. Chem. Res., 1978, 11, 327-333. For the use of polymeric reagents in functional group transformations, see A. Akelah and D. C. Sherrington, Application of Functionalized Polymers in Organic Synthesis, Chem Rev., 1981, 81, 557-587 and W. T. Ford and E. C. Blossey, Polymer Supported Reagents, Polymer supported Catalysts, and Polymer Supported Coupling Reactions, in Preparative Chemistry using Supported Reagents, Pierre Laszlo, ed., Academic Press, Inc., 193-212 (1987). For the use of polymeric reagents in oxidation reactions, see J. M. J. Frechet et al., J. Org. Chem., 1978, 43, 2618 and G. Cainelli et al., J. Am. Chem. Soc., 1976, 98, 6737. For the use of polymeric reagents in halogenation reactions see J. M. J. Frechet et al., J. Macromol. Sci. Chem., 1977, A-11, 507 and D. C. Sherrington et al., Eur. Polym. J., 1977, 13, 73. For the use of polymeric reagents in epoxidation reactions, see J. M. J. Frechet et al., Macromolecules, 1975, 8, 130 and C. R. Harrison et al., J. Chem. Soc. Chem. Commun., 1974, 1009. For the use of polymeric reagents in acylation reactions see M. B. Shambhu et al., Tet. Lett., 1973, 1627 and M. B. Shambhu et al., J. Chem. Soc. Chem. Commun., 1974, 619. For the use of polymeric reagents in Wittig reactions, see S. V. McKinley et al., J. Chem. Soc. Chem. Commun., 1972, 134.
Polymeric reagents also have found widespread use in combinatorial and parallel synthesis and for preparing combinatorial and parallel synthesis libraries. For discussions, see F. Balkenhohl et al., Angew. Chem. Int. Ed. Engl., 1996, 35, 2288-2337; L. A. Thompson et al., Chem Rev., 1996, 96, 555-600; S. R. Wilson and A. W. Czarnik, Combinatorial Chemistry, John Wiley (N.Y.), 1997; D. Obrecht and J. M. Villalgordo, Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries, Elsevier Science Ltd. (UK), 1998.
However, analytical methodology for monitoring and quantifying reactions using polymeric reagents is not as developed as the solid-phase techniques themselves. In general, samples are cleaved from the solid support and analyzed by conventional means, such as TLC, IR and 1H NMR. Removal of samples from the solid support is time consuming and may result in alteration of the reaction product. Therefore, the development of analytical methods for quantitating and monitoring chemical transformations of resin-bound samples is central to the advancement of solid-phase synthetic techniques.
Reported developments related to the analysis of resin-bound samples using fluorine NMR include the use of 19F NMR to characterize products resulting from linking fluorine-containing aromatic compounds to TentaGel resin (Svensson et al., Tetrahedron Lett., 1996, 37, 7649); the use of 19F NMR and magic angle spinning 19F NMR to monitor the nucleophilic displacement of fluorine from 4-fluoro-3-nitrobenzamide linked to Rink resin (Shapiro et al., Tetrahedron Lett., 1996, 37, 4671); the use of fluorinated analogs of p-hydroxymethylbenzoic acid, 3-[4-(hydroxymethylphenyl)]alkanoic acid, and 4-(hydroxymethyl)phenoxyacetic acid linkers for monitoring solid-phase synthesis using gel-phase 19F NMR (Svensson et al., Tetrahedron Lett., 1998, 39, 7193-7196); and a method of quantifying resin loading using gel phase 19F NMR (Stones et al., Tetrahedron Lett., 1998, 39, 4875-4878).
This invention concerns methods for monitoring and quantifying solid-phase reactions using solid-phase synthetic reagents in which one or more fluorine atoms are permanently incorporated as an internal standard, thereby making it possible to directly quantify and monitor resin loading and subsequent solid-phase reactions by 19F NMR.
This invention is also understood to include both single and multiple-step solid-phase reactions. In the latter case, monitoring and quantifying reactions may be accomplished at one or more steps in a synthetic sequence.
Advantages arising from the practice of this invention include: direct observation of reaction yields and kinetics of polymer-supported moieties; rapid sample preparation requiring only washing a solid-phase reaction product to remove soluble 19F labeled species prior to the analysis; high analytical sensitivity due to the high natural abundance of the 19F isotope; large 19F NMR spectral dispersion (about 200 ppm); simple spectra comprising a single resonance for each non-equivalent 19F nucleus; rapid analysis (spectral acquisition typically within  less than 5 minutes) using standard NMR hardware; the method is suited to high throughput analysis.
This invention is further directed, in general, to a method using 19F NMR spectroscopy to quantitate or monitor polymerization reactions and chemical modifications of polymeric compounds. To effect this method, it is necessary that fluorine-containing monomers, reagents or both are employed in the polymerization or chemical modification processes.
In order to calculate the amounts of reagents for use in subsequent reactions and for optimization of the subsequent chemistry, it is necessary to determine the loading of the fluorine-containing solid-phase reaction product.
Accordingly, in its principal aspect, this invention is directed to a method of quantitating a solid-phase reaction, this method comprising the steps of:
(a) reacting a solid-phase reaction component or a fluorine-containing solid-phase reaction component with a reactant or fluorine-containing reactant to form a fluorine-containing solid-phase reaction product;
(b) obtaining a 19F NMR spectrum of the fluorine-containing solid-phase reaction product; and
(c) comparing the integral corresponding to the fluorine-containing solid-phase reaction product 19F resonance to the integral corresponding to a standard 19F resonance.
In another aspect, this invention is directed to a fluorine-containing solid-phase reaction component comprising a known quantity of fluorine, which reaction component is prepared by reacting a solid-phase reaction component with a quantity of a fluorine-containing reactant.